Metabolic dysregulation in the Atp7b-/- Wilson's disease mouse model.

Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b-/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b-/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b-/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b-/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b-/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b-/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b-/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b-/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b-/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b-/- mice.

Considerations for optimizing Wilson's disease patients' long-term follow-up. / Enfermedad de Wilson: consideraciones para optimizar el seguimiento a largo plazo.

Wilson's disease is a sistemic genetic disease caused by the excessive accumulation of copper. The first and main involvement is in the liver, which can range from mild and transient elevation of transaminases to the onset of an overt cirrhosis or acute liver failure. It is known that up to 20-30% of these patients may evolve to liver cirrhosis during follow-up. In clinical practice, liver fibrosis is assessed mainly by using indirect and non-invasive tools (laboratory tests, liver elastography, ultrasound), similar to other prevalent chronic liver diseases. However, despite the fact that liver elastography is a valuable tool in general hepatology, the evidence of its usefulness and accuracy in Wilsons disease is scarce. This review summarizes the available scientific data and their limitations in Wilson's disease.

The dilemma to diagnose Wilson disease by genetic testing alone.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder of hepatic copper excretion. About sixty per cent of patients present with liver disease. WD is considered a fatal disease if undiagnosed and/or untreated but recent data indicate that disease penetrance may not be 100%. MATERIALS AND METHODS: All patients underwent liver biopsy as part of the diagnostic workup. Genetic testing for ATP7B was performed by Sanger sequencing. RESULTS: We report on a large family with multiple affected siblings. The first patient (male, 31 years) underwent orthotopic liver transplantation (OLT) because of fulminant WD. He was homozygous for p.G710A. One asymptomatic brother (37 years) had the same mutation. He is doing well on chelation therapy. Fifteen years later, a second-degree sibling (female, 16 years) presented with fulminant WD and underwent OLT. She was compound heterozygote (p.G710A/p.G710S). Further family screening revealed a third mutation (p.V536A) in a female (21 years) and male (16 years) compound-heterozygote sibling (p.G710A/p.V536A). In both, serum ceruloplasmin and 24-hour urinary copper excretion were normal. Liver biopsy showed normal histology and a quantitative hepatic copper content within the normal range or only slightly elevated (19 and 75 µg/g dry weight, respectively). No decoppering treatment was initiated so far. CONCLUSION: Genetic testing alone is not always sufficient to diagnose WD in asymptomatic patients, and human mutation databases should be used with caution. Even patients carrying two disease-causing mutations do not necessarily have demonstrable alteration of copper metabolism. Asymptomatic siblings diagnosed by genetic screening require further testing before initiating treatment.

Computing the Pathogenicity of Wilson's Disease ATP7B Mutations: Implications for Disease Prevalence.

Genetic variations in the gene encoding the copper-transport protein ATP7B are the primary cause of Wilson's disease. Controversially, clinical prevalence seems much smaller than the prevalence estimated by genetic screening tools, causing fear that many people are undiagnosed, although early diagnosis and treatment is essential. To address this issue, we benchmarked 16 state-of-the-art computational disease-prediction methods against established data of missense ATP7B mutations. Our results show that the quality of the methods varies widely. We show the importance of optimizing the threshold of the methods used to distinguish pathogenic from nonpathogenic mutations against data of clinically confirmed pathogenic and nonpathogenic mutations. We find that most methods use thresholds that predict too many ATP7B mutations to be pathogenic. Thus, our findings explain the current controversy on Wilson's disease prevalence because meta-analysis and text search methods include many computational estimates that lead to higher disease prevalence than clinically observed. As proteins and diseases differ widely, a one-size-fits-all threshold cannot distinguish pathogenic and nonpathogenic mutations efficiently, as shown here. We also show that amino acid changes with small evolutionary substitution probability, mainly due to amino acid volume, are more associated with the disease, implying a pathological effect on the conformational state of the protein, which could affect copper transport or adenosine triphosphate recognition and hydrolysis. These findings may be a first step toward a more quantitative genotype-phenotype relationship of Wilson's disease.

Biochemical and molecular characterisation of neurological Wilson disease.

BACKGROUND: To identify biochemical and genetic features that characterise neurological Wilson disease as a distinct disease subgroup. METHODS: Detailed biochemical profiles and genotypic characteristics of neurological (86 patients) and hepatic subgroups (233 patients) from 368 unrelated Korean families were analysed. RESULTS: Compared with patients in the hepatic subgroup, patients in the neurological subgroup had a later age at onset, a higher proportion with Kayser-Fleischer rings and higher serum creatinine levels, and a lower proportion with favourable outcome (62% vs 80%, P<0.016). At diagnosis, the neurological subgroup had lower serum ceruloplasmin (3.1±2.1 mg/dL vs 4.2±3.2 mg/dL, P<0.001), total copper (26.4±13.8 µg/dL vs 35.8±42.4 µg/dL, P=0.005), free copper (17.2±12.5 µg/dL vs 23.5±38.2 µg/dL, P=0.038) and urinary copper (280.9±162.9 µg/day vs 611.1±1124.2 µg/day, P<0.001) levels. Serum aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and total bilirubin levels, as well as prothrombin time, were also lower in the neurological subgroup. Liver cirrhosis was more common but mostly compensated in the neurological subgroup. Frameshift, nonsense or splice-site ATP7B mutations and mutations in transduction or ATP hinge domains (2.4% vs 23.1%, P=0.006) were less common in the neurological subgroup. CONCLUSION: The neurological subgroup had distinct clinical, biochemical and genetic profiles. Further studies are required to identify the factors, with or without association with copper metabolism, underlying the neurological presentation for which treatment needs to be targeted to improve the clinical outcome of this subgroup.

Copper Inhibits the AlkB Family DNA Repair Enzymes under Wilson's Disease Condition.

Disturbed metabolism of copper ions can cause diseases such as Wilson's disease (WD). In this work, we investigated the inhibitory effect of Cu(II) ion in vitro on the AlkB family DNA repair enzymes, which are members of the Fe(II)/alpha-ketoglutarate-dependent dioxygenase and include human ALKBH2, ALKBH3, and E. coli AlkB proteins. None of the three proteins was significantly inhibited under normal cellular copper concentrations. However, under WD related condition, we observed that the activities of all three enzymes were strongly suppressed (from 95.2 to 100.0%). We also noted the repair efficiency under ds-DNA condition was less susceptible than ss-DNA to the inhibition.

[Mutation analysis of 35 Wilson's disease pedigrees].

OBJECTIVE: To analyze the features of genetic mutations underlying Wilson's disease and provide prenatal and presymptomatic diagnosis. METHODS: For 35 pedigrees affected with the disease, the exons and exon-intron boundaries of the ATP7B gene were amplified with polymerase chain reaction and subjected to Sanger sequencing. After the genotypes of parents of the probands were determined, prenatal diagnosis were performed through chorionic villus sampling. RESULTS: The overall rate for mutation detection was 92.9%. A total of 24 distinct mutations were detected, which included 7 novel mutations, i.e., c.3871G>A(p.A1291T), c.2593_2594insGTCA, c.2790_2792delCAT, c.3661_3663delGGG, c.3700delG, c.4094_4097delCTGT, and IVS6+1G>A. Three mutations, including R778L (c.2333G>T)(45.7%), A874V (c.2621C>T)(7.1%) and P992L (c.2975C>T)(7.1%), were relatively common. Two presymptomatic patients were detected through familial screening, for whom treatment was initiated. Prenatal genetic diagnosis has verified three healthy fetuses and one carrier. CONCLUSION: In this study the most popular mutation ofATP7B gene is R778L and 7 novel mutations have been identified in this gene. For pedigrees of Wilson's disease, genetic counseling in addition with prenatal and presymptomatic diagnosis should be provided through Sanger sequencing and haplotype analysis.

Automation of o-dianisidine assay for ceruloplasmin activity analyses: usefulness of investigation in Wilson's disease and in hepatic encephalopathy.

Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks.

Diverse functional properties of Wilson disease ATP7B variants.

BACKGROUND & AIMS: Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS: We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper ((64)Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS: Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS: Variants in ATP7B associated with Wilson disease disrupt the protein's transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype-phenotype correlation and mechanisms of disease pathogenesis.

Difference in stability of the N-domain underlies distinct intracellular properties of the E1064A and H1069Q mutants of copper-transporting ATPase ATP7B.

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu(1064) and His(1069) is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.

Genetic modifiers of liver injury in hereditary liver disease.

The genetic background of patients with liver diseases modulates hepatic injury, with some individuals being predisposed to better defenses and regenerative capacity. In this review, we focus our description of this phenomenon on inherited disorders affecting the liver, with a particular emphasis on Wilson disease (WD), genetic hemochromatosis, and α-1 anti-trypsin disease (A1-AT). Wide variations in the clinical phenotype of WD may in part be related to the mutations of the ATP7B genotype, though modifier genes and environmental factors also likely play an important role. There is also a significant variability in the expression of iron overload in patients with genetic hemochromatosis that are homozygous for the C282Y mutation. Homozygosity for the A1-ATZ mutation is generally required for the development of liver disease in A1-AT although there is increasing evidence for modifier effects from a heterozygous genotype in other liver diseases.

Long-term follow-up of Wilson disease: natural history, treatment, mutations analysis and phenotypic correlation.

BACKGROUND AND AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism. When treated, the outcome can be excellent, although the long-term survival has yet to be well documented. The aim of this study was to describe the long-term outcome of a cohort of patients with WD and to assess those factors affecting the phenotypic manifestation of WD. METHODS: The presence of mutations to the ATP7B gene, the clinical manifestations, treatments and the long-term outcomes were analysed retrospectively in 117 patients with WD (59 men and 58 women, aged at evaluation 38.5 ± 11, range 16-63 years). RESULTS: Fifty-five patients with a neurological presentation, 51 patients with a hepatic presentation and 11 asymptomatic patients were followed up for an average of 15.1 ± 10 years (median 12 years, range 1-41 years). The H1069Q ATP7B gene mutation was the most frequent genetic variant (54.3%); the frequency of this mutation did not differ between patients with either the hepatic or the neurological presentation (P = 0.099). d-penicillamine or zinc salts (81 and 17% respectively) were used for treatment, and three patients underwent liver transplantation. The majority of symptomatic patients became asymptomatic, or improved, during the follow-up (82% patients with hepatic presentation, 69% with neurological presentation). The long-term survival of patients with WD did not differ from that of the general Czech population (P = 0.95). CONCLUSIONS: Long-term follow-up shows a satisfactory response in the great majority of adequately treated patients with WD and survival coincides with that of the general population.

Distinct clinical courses according to presenting phenotypes and their correlations to ATP7B mutations in a large Wilson's disease cohort.

INTRODUCTION AND AIMS: Wide phenotypic and genotypic heterogeneities in Wilson's disease (WD) have been reported, hampering the study of their correlations. The goal of this study was to identify the factors related to these diversities. METHODS: Clinical courses and molecular genetic characteristics were analysed in 237 unrelated Korean WD families. The average follow-up period was 8.2 ± 5.8 years. RESULTS: Presenting phenotypes were classified as H1 (12.2%), H2 (42.4%), N1 (21.6%), N2 (0.4%), NX (0.4%), presymptomatic (22.4%) and other (0.4%), modifying the guidelines by Ferenci and colleagues. Age at presentation was youngest and cirrhosis was rarest in the presymptomatic group. Decompensated cirrhosis was the highest in the H1 group. Favourable outcome was rarest in the N1 group. Forty-seven (11 novel) ATP7B mutations were identified in 85% of the 474 alleles. Multiplex ligation-dependent probe amplification assays in ATP7B and analyses of ATOX1 and COMMD1 genes identified no additional mutations. Yeast complementation assays demonstrated functional perturbation of the seven novel missense mutants. Five major mutations, p.Arg778Leu, p.Ala874Val, p.Asn1270Ser, p.Lys838SerfsX35 and p.Leu1083Phe, accounted for 63% of the alleles. H1 was more common, age at presentation was younger and N1+N2+NX tended to be less common in patients with nonsense, frame shifting or splicing mutations than in those with missense mutations alone. Patients with both mutations in the transduction (Td) or the ATP hinge domain showed presymptomatic or hepatic manifestations but no neurological manifestation. CONCLUSIONS: The presenting phenotype strongly affects the clinical outcome of WD, and is related to the ATP7B mutation type and location, providing an evidence for genotype-phenotype correlations in WD.

Truncating mutations in the Wilson disease gene ATP7B are associated with very low serum ceruloplasmin oxidase activity and an early onset of Wilson disease.

BACKGROUND: Mutations in the gene ATP7B cause Wilson disease, a copper storage disorder with a high phenotypic and genetic heterogeneity. We aimed to evaluate whether 'severe' protein-truncating ATP7B mutations (SMs) are associated with low serum ceruloplasmin oxidase activities and an early age of onset when compared to missense mutations (MMs). METHODS: The clinical phenotype of 59 genetically confirmed WD patients was analyzed retrospectively. Serum ceruloplasmin was measured by its oxidase activity with o-dianisidine dihydrochloride as substrate and immunologically. RESULTS: Thirty-nine patients had two MMs, 15 had the genotype SM/MM, and 5 patients had two SMs on their ATP7B alleles. Enzymatic and immunologic serum ceruloplasmin levels differed significantly between the three groups (P < 0.001 and P < 0.01, respectively). The lowest levels were measured in patients with two SMs (0.0 U/L; IQR, 0.0-0.0 U/L and 0.02 g/L; IQR, 0.01-0.02 g/L, respectively) and the highest in patients with two MMs (17.8 U/L; IQR, 5.8-35.1 U/L and 0.11 g/L; IQR,0.10-0.17 g/L, respectively). The age of onset was also significantly different between the three patient groups (P < 0.05), with SM/SM patients showing the earliest onset (13 years; IQR, 9-13 years) and patients with two MMs showing the latest onset (22 years; IQR, 14-27 years). By ROC curve analysis a ceruloplasmin oxidase level

Evidence for a critical role of ceruloplasmin oxidase activity in iron metabolism of Wilson disease gene knockout mice.

BACKGROUND AND AIM: Wilson disease is a genetic disorder associated with copper overload due to mutations within the ATP7B gene. Although copper and iron metabolism are closely linked, the influence of mutations of the ATP7B gene on iron homeostasis is unknown. Therefore, the present study was carried out to elucidate iron metabolism in Atp7b(-/-) mice, an animal model of Wilson disease. METHODS: Hepatic iron content, serum iron parameters and blood hemoglobin levels of Atp7b(-/-) mice and wild type mice were studied. Hepatic and duodenal expression of iron metabolism-related genes was measured quantitatively by real-time reverse transcription-polymerase chain reaction and post-translational expression of Dmt1 was analyzed by immunoblot. RESULTS: Atp7b(-/-) mice displayed copper accumulation (P < 0.001), slightly elevated hepatic iron content (P = NS), and a low serum ceruloplasmin oxidase activity (1.5 +/- 1.9 U/L vs 18.9 +/- 4.0 U/L, P < 0.001) when compared with wild type mice. Serum iron, serum transferrin saturation, and blood hemoglobin levels were significantly lower in Atp7b(-/-) mice compared with controls (121.2 +/- 35.3 microg/dL vs 201.8 +/- 34.9 microg/dL (P < 0.001); 44.0 +/- 12.7% vs 68.0 +/- 8.2% (P < 0.001); and 12.7 +/- 0.2 g/dl vs 15.3 +/- 0.1 g/dl (P < 0.001), respectively). Hepatic mRNA expression of hepcidin, TfR-1, TfR-2, hemojuvelin, and Dmt1 + IRE did not differ significantly between Atp7b(-/-) and wild type mice. In the duodenum of Atp7b(-/-) mice Dmt1 + IRE and hephaestin did not show any differences in their mRNA levels compared with wild type mice, while Dcytb mRNA expression was 1.7-fold increased compared with wild type mice (P = 0.01). CONCLUSION: Atp7b(-/-) mice demonstrated decreased serum iron parameters and hemoglobin levels most likely related to a low serum ceruloplasmin oxidase activity and not due to total body iron deficiency.

[Wilson's disease]. / Choroba wilsona.

Wilson's disease is caused by a P-type ATP-ase gene mutations with reduced biliary copper excretion and accumulation copper in the liver and other tissues. Clinical symptoms can be heterogeneous but in many cases on the first stage the only abnormalities is elevation of aminotransferase activity. In some cases the first fatal symptom of disease is acute liver failure, therefore early diagnosis and treatment is essential. We present an actual recommendations for diagnosis and treatment of patients with Wilson's disease.

Solution structures of the actuator domain of ATP7A and ATP7B, the Menkes and Wilson disease proteins.

ATP7A and ATP7B are two human P(1B)-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorylated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fold of the domain (such as Gly860Val in ATP7A) or affect the network of communication within the domain (such as Leu873Arg in ATP7A) or with the other domains of the enzyme (such as Gly853Arg in ATP7A).

Tracing copper-thiomolybdate complexes in a prospective treatment for Wilson's disease.

Wilson's disease is a human genetic disorder which results in copper accumulation in liver and brain. Treatments such as copper chelation therapy or dietary supplementation with zinc can ameliorate the effects of the disease, but if left untreated, it results in hepatitis, neurological complications, and death. Tetrathiomolybdate (TTM) is a promising new treatment for Wilson's disease which has been demonstrated both in an animal model and in clinical trials. X-ray absorption spectroscopy suggests that TTM acts as a novel copper chelator, forming a complex with accumulated copper in liver. We have used X-ray absorption spectroscopy and X-ray fluorescence imaging to trace the molecular form and distribution of the complex in liver and kidney of an animal model of human Wilson's disease. Our work allows new insights into metabolism of the metal complex in the diseased state.